Med. Weter. 68 (6), 359-361, 2012

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Borzym E., Maj J., Matras M.
Impact of organic fixatives on the stability of the DNA genetic material of the koi herpes virus (KHV)
Studies to identify the koi herpes virus using the PCR are continuously developed and introduced into routine diagnostics. Well-selected ways of storing tissues that are later used for DNA extraction and for the amplification reaction are critical points in methods using molecular biology techniques. The objective of the studies was to determine the impact of the fixating properties of the two most frequently used organic compounds, ethanol 96% and 80%, and isopropanol, on the stability of the nucleic acid isolated and on the preservation of the koi herpes virus in selected tissues and organs of carps. The experimental fish were infected via a bath in a small 20 l aquarium, where 5 ml of a KHV reference strain suspension had been added to the water. The presence and stability of the total DNA isolated from tissues and organs (skin, brain, kidney, gills) was checked using PCR with TK primers designed by Bercovier, and a product of 409 bp was obtained. Primers amplifying a gene fragment of beta-actin, which is found in smooth muscles and non-muscle cells of fish, were used as isolation control in multiplex PCR. On the basis of the studies it was observed that just on the day the samples were taken from the internal organs of fish and a month later, when they were collected from the archived organs, a product of 409 bp and 259 bp was obtained in all the samples analysed. The best organic fixative of organs and tissues sampled for molecular tests to identify KHV is ethanol 96%, even after half a year of archiving. Ethanol 80% and isopropanol also ensure sufficient fixation for up to a month. The most stable virus DNA is isolated from fish skin and gills.
Key words: koi herpesvirus (KHV), tissue storage, organic fixatives, DNA extraction